Reference is made to copending application Ser. No. 07/103,237, filed Sep. 30, 1987, now abandoned, entitled "Novel Bidentate Conjugate and Method of Use Thereof", which is filed concurrently herewith in the names of Paul Harris and Chan S. Oh.
1. Specific Binding Assays
Methods for measuring immunochemical or other types of specific binding reactions have become widely accepted in the field of medical testing in recent years. Generally speaking, an immunochemical reaction involves the reaction between at least one antigen and at least one antibody. An antigen is ordinarily a substance, such as a protein or carbohydrate, which is capable of inducing an immune response; i.e., antibody production, when introduced into an animal or human body. The antibodies produced as a result of the immune response are bivalent in nature, generally being depicted as a "Y", wherein each arm of the "Y" is capable of binding to the antigen which induced production of the antibody. The presence of a particular antigen or antibody in a patient's test sample may indicate a disease state or a bodily condition, such as pregnancy. An immunochemical reaction is one type of specific binding reaction.
Antibody fragments are often used in addition to or in place of whole antibodies in an immunoassay. Generally, there are three different types of antibody fragments. The first type of fragment is designated as either Fab, or F(ab), and is a single arm of the antibody which has been directly cleaved from the whole antibody, usually through digestion by the enzyme papain. Each Fab fragment is monovalent, and has a molecular weight of about 50,000 Daltons, compared to the approximate 150,000 Dalton size of the whole antibody. The second type of fragment is designated as F(ab').sub.2, and consists of both antibody arms, still linked together, but minus the tail which is removed by pepsin digestion. The divalent F(ab').sub.2 fragment has a molecular weight of about 100,000 Daltons, and can be further cleaved into two separate monovalent Fab' fragments (the third type of antibody fragment), also designated as F(ab'), each having a molecular weight of about 50,000 Daltons.
The site on the antigen to which an arm of the antibody binds is referred to as an epitope. Most antigens are polyepitopic, having multiple, and often repeating, binding sites for antibodies. It is the polyepitopic nature of antigens and the bivalent character of antibodies, including F(ab').sub.2 fragments, which enable large antibody:antigen complexes of varying sizes, otherwise known as immunocomplexes, to be formed in an immunoassay.
One particular type of immunoassay which takes advantage of this feature is the sandwich immunoassay, wherein a ternary immunocomplex is formed. The most common type of sandwich immunoassay employs a first insolubilized antibody, usually bound to a solid support, and a second labeled antibody. Each antibody is specific for the antigen of interest (i.e., the analyte to be measured) and binds to a different epitope on the antigen. Preferably, the first antibody binds to an epitope which is remote from the epitope to which the second antibody binds. A ternary complex of insoluble antibody:antigen:labeled antibody is formed where the antigen of interest is contacted with the first and second antibodies. Because each antibody is required to bind to only one antigen, all three types of antibody fragments may be used in this type of method. The presence or absence of the antigen of interest is indicated by the presence or absence of the labeled antibody on the solid support. Ordinarily, the insolubilized phase of the reaction must be separated from the liquid phase in order for either the bound or free labeled antibody to be quantified. Such a reaction is referred to as a heterogenous type of reaction, due to the required separation step.
Nephelometry and turbidimetry require the formation of large aggregates of, e.g., antibody and antigen. Because each antibody must bind to two different antigen molecules, the monovalent Fab and Fab' fragments are generally ineffective in these methods. The large aggregates cause a change in the light scatter of the solution, and are capable of measurement by nephelometric or turbidimetric methods. These methods do not require the use of traditional labels, such as enzymes, radioactive isotopes, fluorescent, or chemiluminescent compounds, to detect the amount of complex formed. Rather, nephelometric and turbidimetric methods directly measure the amount of complex formed. Because no separation step is required, nephelometry and turbidimetry are referred to as homogenous immunoassays.
The multiepitopic nature of the antigen and bivalent character of the antibody will, depending on the amount of antigen and/or antibody present, allow the formation of antigen:antibody complexes large enough to scatter light. Ordinarily, an excess of antibody is used in conjunction with a finite amount of antigen obtained from, e.g., a patient's blood, serum, cerebrospinal fluid (CSF), or urine sample. In such a case, the amount of antigen present in the sample will be the limiting factor in determining the amount and size of antigen:antibody aggregates formed.
In turbidimetry, the reduction of light transmitted through the suspension of particles, or aggregates, is measured. The reduction is caused by reflection, scatter, and absorption of the light by the aggregates. In nephelometry, it is the light scattered or reflected toward a detector that is not in the direct path of light which is measured. In both turbidimetry or nephelometry, the rate of change in light scatter may also be measured as an indication of the amount of antigen present.
Nephelometric procedures have become a convenient method for monitoring antigen:antibody reactions at an early stage, by detecting the rate of growth of complexes capable of scattering light before the complexes separate out of solution as immunoprecipitates. The growth of these complexes begins as a buildup of aggregates which ultimately become large enough to function as "scattering centers". Sternberg, J. C., A Rate Nephelometer for Measuring Specific Proteins by Immunoprecipitation Reactions, Clin. Chem., 23:8, 1456-1464 (1977). The formation of scattering centers can be accelerated by the use of hydrophilic nonionic polymers, such as dextran or polyethylene glycol, which increase the probability of protein-protein interaction by excluding a significant fraction of water. The use of polymers in an immunonephelometric assay also gives the advantages of increased sensitivity and less antiserum consumption.
2. Nephelometric Inhibition Immunoassays for Haptens
Haptens pose a unique problem in immunoassay methods. Haptens are relatively small monovalent molecules, sometimes regarded as incomplete or fragmentary antigens. One common class of haptens is drugs. Theophylline, for example, is a member of this particular subclass of haptens. A hapten is, in and of itself, incapable of inducing an immune response in a human or animal body. This is because haptens are generally too small to be recognized by the body's immune system. However, when coupled to a carrier, such as a protein, the hapten:carrier protein conjugate acts as an antigen which is large enough to induce antibody production. In this way, antibodies can be raised against a hapten. Unlike the relatively large antigens, however, the small hapten molecule is not itself multiepitopic. For this reason, haptens are incapable of forming large complexes or agglomerates with the antibody which has been produced against the hapten.
Consequently, in order to perform nephelometric or turbidimetric assays for haptens, such as in therapeutic drug monitoring, a technique known as nephelometric inhibition immunoassay (NIIA) has been developed, wherein the hapten acts as an inhibitor to complex formation. In traditional NIIA, a second conjugate known as a "developer antigen" is used to develop complexes of sufficient size to cause detectable light scattering. The developer antigen is formed from a second carrier, also usually a protein, conjugated to a multiplicity of hapten molecules. In this way the developer antigen acts as a "polyvalent hapten" which is capable of aggregating with more than one antibody molecule to ultimately form scattering centers. The second carrier protein is sometimes referred to as the "label". The monovalent free hapten present in a patient's test sample acts to inhibit the amount of developer antigen:antibody complexing, by binding to one or both arms of the antibody molecule, thereby reducing complex formation and diminishing the amount of light scatter. Because of the nature of the inhibition immunoassay, both the amount and the rate of the increase detected in light scatter are inversely proportional to the amount of hapten present in the patient's sample.
Several problems have been encountered with prior art turbidimetric or nephelometric inhibition immunoassays. One problem concerns the developer antigen reagent. The traditional developer antigen is generally unstable and requires special storage conditions. The requirement for special storage conditions arises from the fact that the carrier protein of the developer antigen, being a natural proteinaceous substance, degrades relatively rapidly during manufacture as well as during storage. At room temperature, a typical developer antigen can be expected to last only about eight hours. Even at refrigeration temperatures, most developer antigens exhibit a shelf life of only about six months. This greatly compounds the problems of manufacture and distribution and adds to the cost of such products. Moreover, because the carrier protein for the developer antigen is derived from natural sources, considerable variation is encountered in the properties of these proteins. The traditional developer antigen reagent must be carefully prepared, purified, and characterized to insure uniform reactivity. This characterization process is the most expensive aspect of the manufacture of prior art developer antigens.
Prior art NIIA's have also been found to possess limited sensitivity in relation to other types of immunoassays, such as the sandwich immunoassay. This sensitivity limitation results primarily from the scatter caused by other components of the serum sample. For this reason, a test sample must be diluted significantly before being added to the reaction medium of an NIIA, thereby also diluting the concentration of analyte in the reaction medium. In other types of immunoassays, such as the sandwich immunoassay, about 100-200)L of sample are typically added to the reaction medium. In contrast, only about 1-3)L of sample are ordinarily injected into the reaction medium for an NIIA. One method that has been suggested for improving sensitivity involves optimizing the hapten:carrier ratio of the developer antigen, as disclosed in U.S. Pat. No. 4,604,365. High and low hapten:carrier ratios have been reported to result in moderate sensitivity, with improved sensitivity being observed at intermediate ratios. This method, however, is time consuming and fails to show marked increases in NIIA sensitivity.
Yet another problem encountered with the prior art NIIA's involves a phenomenon known as "nonproductive binding". Nonproductive binding occurs, for example, where the two binding arms of the same antibody bind to two hapten moieties on the same developer antigen. In such an instance, there can be no cross-linking with other developer antigens, because there is no free arm on the antibody to bind with another developer antigen. This results in the inefficient use of expensive antibody and developer antigen reagents.
Due to the ease and convenience of the homogenous turbidimetric and nephelometric inhibition immunoassays for haptens, it would be advantageous to have a stable developer antigen which can readily be manufactured to possess consistent characteristics and which exhibits a long shelf life at room temperatures. It would also be advantageous to improve the sensitivity of the NIIA and to reduce the occurrence of nonproductive binding.
3. Prior Art Bifunctional Conjugates
There are several small molecule bifunctional conjugates which exist in the prior art. What is meant by the term "small molecule bifunctional conjugate" is a conjugate which employs two small molecules which are linked together through a spacer moiety. The spacer moiety may be so small as to comprise only one chemical bond (i.e., zero atoms in the spacer). Generally, these molecules are on the order of about 7,000 Daltons or smaller in size. Both molecules act as small molecule ligands and, as such, are each capable of interacting with a substance having a specific binding affinity for the small molecule; i.e., its specific binding partner. This definition specifically excludes conjugates which employ one or more large molecules and/or conjugates which employ one or more chemical moieties which do not have a specific binding partner. For example, the typical enzyme labeled antibody in a sandwich immunoassay is excluded for both reasons; i.e., the antibody moiety is a large macromolecule, generally greater than about 150,000 Daltons in size, and the enzyme moiety, although it acts on a substrate, is not generally considered to be the specific binding partner for the substrate. Also excluded are heterobifunctional cross-linking agents which utilize two chemically reactive groups, rather than two small molecule ligands, one at each end of the conjugate.
There are two classes of small molecule bifunctional conjugates existing in the prior art. The first class of conjugates, known as the homobifunctional conjugate employs identical chemical moieties at each end of the conjugate. The homobifunctional conjugates are generally designed to bring together, or unite, the identical specific binding partner with which each chemical moiety interacts. Where the specific binding partner is polyvalent, large aggregates may be formed.
For example, a Bis-AND homobifunctional conjugate has been proposed as a precipitating agent for enzymes. Larsson, P. and Mosbach, K., Affinity Precipitation of Enzymes, Elsevier/North-Holland Biomedical Press, 98(2), 333-330 (1979). The Bis-NAD conjugate, comprising two NAD moieties separated by a 17 .ANG. spacer moiety, is capable of precipitating the enzyme lactate dehydrogenase (LDH) out of solution by specifically binding to a large LDH molecule at each end of the Bis-NAD. Because each large LDH molecule has multiple binding sites for NAD, large aggregates, similar to those formed in nephelometry, can be obtained. These large aggregates precipitate out of solution carrying along the enzyme. Similar uses of Bis-nucleotides of varying spacer lengths have also been proposed.
Another example of a homobifunctional conjugate which has found application in the prior art is the Bis-biotin conjugate used to examine the structure of avidin. Green, N. M., Konieczny, L., Toms, E. J., and Valentine, R. C., The Use of Bifunctional Biotinyl Compounds to Determine the Arrangement of Subunits in Avidin, Biochemistry, 125, 781-791 (1971). Where the two biotin moieties of the Bis-biotin conjugate were joined by a spacer moiety of approximately 18/, strong complexes or polymers were formed with the multivalent macromolecule avidin.
The second class of prior art small molecule bifunctional conjugates is the heterobifunctional conjugate. In contrast to the homobifunctional conjugate, the heterobifunctional conjugate employs a different chemical moiety at each end of the conjugate. Each of these chemical moieties is capable of interacting with a different specific binding partner. These prior art heterobifunctional conjugates have been used almost exclusively as modulators, wherein the binding of a specific binding partner to one of the chemical moieties hinders or precludes the simultaneous binding of the corresponding specific binding partner to the other chemical moiety. Simultaneous binding at both ends of the heterobifunctional conjugate is precluded by steric hindrance, generally caused by the use of shorter spacer lengths than those required to achieve the desired simultaneous binding where homobifunctional conjugates are employed as described above. In other words, the binding of a macromolecular specific binding partner to the modulator moiety of the conjugate sterically inhibits the binding of the specific binding partner to the chemical moiety responsible for producing signal.
The prior art heterobifunctional conjugates generally employ a small molecule ligand of interest, usually an analyte, as one of the chemical moieties of the conjugate. This chemical moiety can compete with free analyte, such as from a test sample, for a limited amount of specific binding partner for the analyte. The other chemical moiety of the heterobifunctional conjugate is usually a "surrogate" label such as an enzyme modulator or a prosthetic group or other cofactor for an enzyme. The surrogate label modulates the activity of the indicator label, usually an enzyme. These types of prior art heterobifunctional conjugates are generally of use in homogenous enzyme immunoassays, because the degree of activity of the enzyme is directly influenced by the antigen:antibody reaction. No separation step is required to determine the amount of enzyme activity attributable to the bound enzyme versus the activity attributable to the free enzyme, as in heterogenous enzyme immunoassays.
The enzyme modulated immunoassay is based on the ability of the small molecule ligand:enzyme modulator heterobifunctional conjugate to influence the activity of the indicator enzyme. See, for example, U.S. Pat. No. 4,134,792, which also discloses larger surrogate labeled conjugates. In this instance, the spacer moiety between the ligand moiety and the enzyme modulator moiety is relatively short, preferably being on the order of about 1-10 carbon atoms or heteroatoms in length; i.e., about 1.3 to about 14.0/.
The small molecule ligand:enzyme modulator heterobifunctional conjugate competes with ligand from a test sample for a limited amount of antibody. If the small molecule ligand:enzyme modulator heterobifunctional conjugate is bound to the antibody; i.e., through the ligand moiety of the conjugate, the enzyme modulator cannot affect the activity of the indicator enzyme. Modulators which increase or decrease the enzyme activity of the indicator enzyme can be used, although modulators which decrease enzyme activity; i.e., enzyme inhibitors, are more commonly used. In assays employing an inhibiting modulator, the observed enzyme activity will be inversely proportional to the concentration of analyte.
A similar type of homogenous enzyme immunoassay is based on the use of a small molecule ligand:enzyme cofactor heterobifunctional conjugate. In a broad sense, an enzyme cofactor operates as a positive enzyme modulator; i.e., a modulator which increases enzyme activity. Generally, enzymes may be divided into two groups: (1) enzymes where enzymatic activity is due solely to the protein nature of the enzyme; and, (2) enzymes where optimal enzymatic activity is dependent on a heat-stable, non-protein structure called a cofactor. Immunoassays employing enzymes of this second group lend themselves to modulation through the use of a small molecule ligand:enzyme cofactor heterobifunctional conjugate.
Cofactors vary in nature from simple inorganic ions to more complex organic materials, many of which are derivatives of vitamins, such as biotin and flavin adenine dinucleotide (FAD). The organic cofactors are often referred to as coenzymes. In certain cases, as is typical with prosthetic groups, the cofactor is firmly bound, usually through a covalent linkage, to the protein moiety of the parent enzyme which is otherwise individually known as the apoenzyme. In the classical jargon of enzymology, the complete, enzymatically active enzyme:cofactor complex is termed a holoenzyme.
Residues of certain cofactors such as FAD, flavin mononucleotide (FMN), or heme, for example, provide particularly good enzyme prosthetic groups for use in a small molecule ligand:enzyme prosthetic group heterobifunctional conjugate. See, for example, U.S. Pat. No. 4,238,565, which also discloses larger surrogate labeled conjugates. In this case, the spacer moiety between the ligand moiety and the enzyme prosthetic group moiety is no more than 14 carbon atoms, and more commonly 1-6 carbon atoms or 0-5 heteroatoms in length; i.e., about 1.3 to about 14.0/.
According to U.S. Pat. No. 4,238,565, the ligand:prosthetic group heterobifunctional conjugate (for example ligand:FAD) competes with the ligand in a test sample for a limited amount of antibody. If the ligand:FAD conjugate is bound by the antibody, it can no longer combine with the apoenzyme to form an enzymatically active holoenzyme. The observed enzyme activity is directly related to the concentration of analyte present in the test sample.
The one exception to this modulator type of use of the heterobifunctional conjugate is in the area of column chromatographic purification. A substance may be purified by passing a solution containing the substance through a chromatographic column in one of two ways. In one manner of purification, the column contains attached groups that specifically bind to or otherwise pull specific impurities from the solution. In an alternate manner of purification, groups which specifically bind to the substance sought to be purified are immobilized on the column. These groups pull the desired substance out of solution. In the latter case, the substance must later be eluted from the column.
General ligand affinity chromatography follows the latter approach and is based on the principle that a single immobilized ligand is able to adsorb a family of enzymes, such as dehydrogenases or kinases, with the isolated enzyme being subsequently eluted under conditions favoring biospecific elution. Often a cofactor or cofactor fragment is used as the general ligand.
The insolubilized small molecule heterobifunctional conjugate AMP-ATP has been proposed for use in general ligand affinity chromatography. Lee, C.-Y., Larsson, P. O., and Mosbach, K., Synthesis of the Bifunctional Dinucleotide AMP-ATP and its Application in General Ligand Affinity Chromatography, J. Solid Phase Biochem., 2(1), 31-39 (1977). The ATP moiety (specific for kinases) is attached to a Sepharose.RTM. 4B (cross-linked agarose gel, Pharmacia, Uppsala, Sweden) column through a previously bound AMP moiety (specific for dehydrogenases). It has been reported that the ATP and AMP moieties retain their affinity behavior toward kinases and dehydrogenases, respectively, even when both are bound to the Sepharose.RTM. column through the AMP moiety. An attempt to prepare a soluble AMP-ATP dinucleotide has proved unsuccessful. Id.
None of these prior art bifunctional conjugates has been applied to nephelometric or turbidimetric assay procedures. Moreover, these prior art bifunctional conjugates lack the versatility and sensitivity that could be achieved with, e.g., a trifunctional conjugate. For example, the small molecule homobifunctional conjugate is useful only for linking up like molecules, while the small molecule heterobifunctional conjugate is limited in application to only certain types of assays which lend themselves to modulation by such a conjugate. It would be advantageous to have a trifunctional conjugate capable of agglomerating dissimilar macromolecules as well as serving a modulating function in a greater variety of immunoassays.
4. The Use of Avidin and Biotin in Immunoassays
Avidin and biotin are both naturally occurring compounds. Avidin is a relatively large macromolecular protein and is found in egg whites. Avidin contains four subunits. Biotin is a relatively small, stable, water-soluble vitamin. Each of the four subunits of an avidin. molecule is capable of specifically binding to a molecule of biotin. The binding reaction between avidin and biotin is very strong, with the binding constant being approximately 10.sup.15 L/mole. The very strong nature of this bond has been found to persist even when biotin is conjugated, by means of its carboxyl group, to another molecule, or when avidin is attached to another molecule. When biotin is conjugated to another molecule, the resulting conjugate is usually referred to as a biotinylated compound; e.g., a biotinylated protein. A biotinylated protein may, for example, quickly become strongly bound to a corresponding avidin-attached molecule. This feature of linking up biotinylated compounds with avidin conjugates has been employed, with varying degrees of success, mostly in hererogenous immunoassays.
Two such applications pertain to sandwich immunoassays. In one instance, the avidin:biotin bond is utilized at the label end of the sandwich. This is seen in U.S. Pat. No. 4,228,237, wherein a biotinylated specific binding partner for the ligand to be measured is employed in conjunction with enzyme-labeled avidin. In another instance, the biotin:avidin bond may be used at the insolubilized end of the sandwich formed in a sandwich immunoassay. For example, U.S. Pat. No. 4,298,685 teaches the use of insolubilized avidin which is ordinarily added after the labeled sandwich has been formed in solution. Where the unlabeled antibody of the sandwich has previously been tagged with biotin, the insolubilized avidin is able to capture the labeled sandwich from the solution. These applications are not applicable to nephelometry or turbidimetry. Moreover, the additional conjugation steps required for preparing reagents makes such methods less attractive economically.
Avidin has also been used in homogenous immunoassays as the enzyme modulator label component of a larger surrogate labeled conjugate, which is used in a manner similar to the previously discussed small molecule ligand:enzyme modulator heterobifunctional conjugates. Avidin is the natural inhibitor of biotin-containing enzymes such as pyruvate carboxylase. When the biotin moiety of these enzymes is tied up, i.e., complexed with avidin, the activity of the enzyme ceases or is diminished. This is because biotin is a required cofactor of these enzymes, and, where the biotin moiety is incapable of functioning as a cofactor, enzyme activity is inhibited. Avidin may thus be used as a modulator label, due to its ability to modulate or control the activity of biotin-containing enzymes which, when allowed to act upon a substrate, yield a measurable signal in certain homogenous immunoassay systems.
U.S. Pat. No. 4,550,075 discloses avidin as the modulator label component of a larger labeled conjugate for use in a homogenous immunoassay. The labeled conjugate of U.S. Pat. No. 4,550,075 takes advantage of the large molecular size of avidin, which, at approximately 63,000 Daltons, is considerably larger than most modulator labels; i.e., enzyme inhibitors. This enables avidin to alleviate a steric hindrance problem typically encountered with larger surrogate labeled conjugates. For example, where a small molecule ligand:enzyme modulator heterobifunctional conjugate is used, the relative small size of the typical low molecular weight enzyme modulator is comparable to that of the ligand portion of the conjugate, and the modulator is therefore able to function effectively in the assay. Where, however, the ligand is much larger than the usual enzyme modulator, such as where the ligand is an antigen, the typical enzyme modulator is dwarfed by the size of the ligand, and the activity of the modulator label is sterically inhibited even in the absence of binding by the ligand component to its specific binding partner.
This steric hindrance problem has been addressed to some extent in the previously cited U.S. Pat. No. 4,238,565, wherein it is suggested that a slightly longer spacer moiety be employed where the ligand is a larger molecule of relatively high molecular weight. In any event, the spacer moiety may not exceed about 14 carbon atoms and 0-5 heteroatoms in length. The objective is that steric hindrance should occur only when the ligand moiety of the conjugate is bound to its specific binding partner, but not while the ligand moiety of the conjugate is free. U.S. Pat. No. 4,550,075, on the other hand, simply takes advantage of the inability of large ligands, such as antigens, to sterically hinder the activity of the macromolecular enzyme modulator avidin. Steric hindrance occurs only when the ligand moiety is bound to its specific binding partner.
The avidin:biotin bond has not been made of use in nephelometric or turbidimetric procedures, although the high specificity and strong nature of the bond would seemingly make it desirable in such procedures. The only use of avidin:biotin in complex formation is the previously noted use of Bis-biotin to agglomerate avidin. Likewise, avidin has not been used to create a desired steric hindrance, but, instead, to avoid steric hindrance where the analyte member of a bifunctional conjugate for use in a modulated assay is a macromolecular antigen. It would be desirable to take advantage of the steric hindrance-inducing ability of the macromolecular specific binding partner avidin, particularly in the area of NIIA's and modulated assays.
5. Prior Art Proximity Assays
There exist in the prior art several types of immunoassays wherein a measurable interaction occurs between the labeled portion of a labeled antigen and the labeled portion of a labeled antibody when the two labels are brought into close proximity with each other; i.e., pursuant to a specific binding reaction between the antigen and the antibody. These immunoassays may be referred to as "proximity assays" because they require that the labels be proximate to each other before a measurable reaction can occur. Where the proximity is caused by the binding of a labeled antigen or hapten, such as an analyte of interest, to a labeled antibody, the signal obtained from the interaction between the proximately located labels can be correlated to the amount of antigen or hapten present in a test sample. Most assays employing proximity labels are competitive assays wherein the amount of signal generated bears an inverse relationship to the amount of analyte present in a sample.
One type of proximity assay, known as an "enzyme channeling" assay, employs as labels an enzyme pair from a multienzyme complex. Multienzyme complexes occur frequently in nature and consist of two or more enzymes that are involved in a sequence of reactions. In other words, the product of one enzyme serves as a substrate for a second enzyme. The product of the second enzyme may serve as the substrate for a third enzyme, and so forth. The enzyme channeling assay utilizes two enzymes which operate in sequence in a multienzyme complex. For convenience, the two enzymes are referred to as a first enzyme and a second enzyme, with the product of the first enzyme serving as a substrate for the second enzyme.
One example of an enzyme channeling assay utilizes hexokinase (HK) and glucose-6-phosphate dehydrogenase (G6PDH) as the first and second enzymes, respectively. Litman, D. J., Hanlon, T. M., and Ullman, E. F., Enzyme Channeling Immunoassay: A new Homogenous Enzyme Immunoassay Technique, Anal. Biochem., 106, 223-229 (1980). The first enzyme (HK) is attached to a finite amount of antibody to the antigen of interest. Both the second enzyme (G6PDH) and antigen identical or analogous to the analyte of interest are bound to microporous beads. In the absence of free antigen, contributed by a patient's test sample, a "channeled system" will exist wherein all of the antibody-bound first enzyme will be bound to the bead through the previously bound antigen. In an "unchanneled system" all of the first enzyme will remain free. This occurs where sufficient free antigen, contributed by test sample, competes so effectively with the bound antigen for a limited amount of enzyme-labeled antibody that none of the enzyme-labeled antibody can bind to the bead.
The amount of enzyme-labeled antibody bound to the bead is a direct function of the amount of free antigen present in a test sample, and can be correlated to the degree of channeling obtained in a particular system. The degree of channeling, or "channeling efficiency", is ordinarily detected by measuring the amount of product generated by the second enzyme. This product is generated only where the second enzyme is able to act on the product generated by the first enzyme in close proximity to the second enzyme.
For example, where HK is the first enzyme, its reaction product, glucose-6-phosphate, will be acted upon by the bound second enzyme, G6PDH, where the HK-labeled antibody is also bound to the bead. In this instance, the glucose-6-phosphate is generated within the vicinity of a high local concentration of G6PDH, such that the G6PDH is able to act on the glucose-6-phosphate before it escapes into the bulk solution. The "channeling efficiency" of the system is the amount of first enzyme product converted by the second enzyme before the product escapes into bulk solution and is an inverse measure of the amount of analyte present in a test sample.
Another type of proximity assay utilizes a phenomenon known as energy transfer. In an energy transfer proximity assay, the measured interaction is usually a change or shift in light emission, which is caused by the transfer of light, or energy, from one label to a second proximately located label. The label from which the energy is transferred is referred to as the "donor label", while the label to which the energy is transferred is referred to as the "acceptor label".
One particular energy transfer assay employs chemiluminescent-labeled biological ligands, such as immunoglobulin G (IgG) and cyclic AMP (cAMP), as the donor labels and their respective fluorescent-labeled antibodies as the acceptor labels. Patel, A., Davies, C. J., Campbell, A. K., and McCapra, F., Chemiluminescence Energy Transfer: A New Technique Applicable to the Study of Ligand-Ligand Interactions in Living Systems, Anal. Biochem., 129, 162-169 (1983). A chemiluminescent compound emits light as the result of a chemical reaction. This particular energy transfer assay takes advantage of the fact that all or a portion of the light, or energy, produced by a chemiluminescent label can be transfered to a fluorescent label, such as fluorescein, where the fluorescent label is brought into close proximity with the chemiluminescent label. The proximity is caused by the specific binding reaction between a chemiluminescent-labeled ligand and its fluorescent-labeled specific binding partner.
Absorption, by the fluorescent label, of energy produced by the chemiluminescent label generally results in a decrease in light emission between about 460 and 487 nm and an increase in light emission between about 525 and 555 nm. Id. The exact wavelength ranges wherein a shift is observed will depend upon the particular chemiluminescent and fluorescent compounds selected as labels.
In a typical competitive binding assay of this type, free analyte from a test sample competes with chemiluminescent-labeled analyte for a finite amount of available fluorescent-labeled antibody. Energy transfer occurs only where the labeled analyte is bound to the labeled antibody. The amount of shift is inversely proportional to the amount of free analyte present in the test sample.
It would be advantageous to have a reagent for competitive proximity assays which would yield a direct positive correlation, rather than an inverse relationship, to the amount of analyte present in a test sample. It would also be advantageous to have reagents which exhibit improved stability characteristics for use in proximity assays. Although chemiluminescent-labeled antigens are reportedly stable for nine months, this stability requires storage at -20.degree. C. Id. It would be desirable if such a reagent were comparably stable at room temperature.
6. Prior Art Conjugation Methods
Most specific binding assays require the use of conjugates of one form or another. For example, the typical sandwich immunoassay requires the conjugation of a label, such as an enzyme or fluorescent compound, to an antibody which functions as the labeled antibody of the sandwich. Conjugates are also used in other processes including synthesis reactions.
A conjugate is simply two substances coupled together. Usually at least one of the substances is a protein. In some cases, such as with an enzyme-labeled antibody, both substances are proteins. Most proteins, as well as certain other substances, have active sites, some or all of which may be important to the ultimate desired performance of the conjugate. Examples of active sites include the active site(s) of an enzyme, the binding arms of an antibody, and the epitope(s) of an antigen or hapten. In conjugating a protein or other substance having active sites it is important to perform the conjugation; i.e., chemical modification, away from the active site.
Conjugation methods generally employ relatively harsh conditions to effect the necessary chemical modification of a protein. This can cause denaturization and/or deactivation of the protein. Moreover, these methods are nondiscriminatory in nature, seeking out a particular type of reactive site on a protein regardless of whether it occurs at or near an active site of the protein. The most common reactive sites used in protein conjugation are amino groups and carboxyl groups, although surface sulfhydryl groups are also frequently used. The random nature of these reactions poses a problem with proteins which have these particular reactive groups at or near the active site.
One method which has been suggested to alleviate the problem of harsh reaction conditions is the use of azide (N.sub.3) as one member of a heterobifunctional cross-linking agent. As previously noted, heterobifunctional cross-linking agents are to be distinguished from small molecule heterobifunctional conjugates, inasmuch as the former utilize two chemically reactive groups, rather than two small molecule ligands. The azide moiety of the cross-linking agent readily converts to an activated nitrene form upon exposure to ultraviolet light. The activated nitrene can then insert into almost any chemical bond without the use of harsh reaction conditions.
Specifically, a heterobifunctional cross-linking agent employing a succinimide ester of a carboxylic acid residue as one chemically reactive group and an azide residue as the other chemically reactive group has been suggested for use in the conjugation of two proteins. Guire, P., Fliger, D. and Hodgson, J., Photochemical Coupling of Enzymes to Mammalian Cells, Pharm. Res. Com., 9(2), 131-141 (1977). An excess of the cross-linking agent is initially added to a solution containing a first protein. The succinimide ester of the carboxylic acid residue binds to any amino functional group of the first protein in a nondiscriminatory manner. This reaction is carried out in the dark due to the reactive nature of the azide residue. Once the reaction between the succinimide ester and the amino groups has taken place and the excess cross-linking agent is eliminated, a second protein is added, and the reaction mixture is exposed to ultraviolet light which converts the azide residue to nitrene. The nitrene is extremely reactive and, in this reactive form, will insert into any chemical bond of the second protein which is readily available to the activated nitrene end of the cross-linking agent. Although relatively mild reaction conditions are encountered in this second phase of the conjugation, relatively good recovery is achieved only where the first protein does not have a reactive free amino group critical to the active site of the protein. In any event, excellent recovery is precluded by the random nature of the nitrene insertion.
Yet another conjugation method addresses the issue of site specificity, but fails to alleviate the problem of harsh reaction conditions. This method targets the polysaccharide moiety of a protein for use as the conjugation site. Most proteins contain a surface polysaccharide group which is located at a site distant from the active site of the protein, making the polysaccharide moiety an ideal location for modification. The active site(s) of a protein does not contain these polysaccharide moieties. Thus, there is little danger of chemically modifying the active site(s). Modification of a polysaccharide moiety generally involves the reactivity of the cis-diol group of the sugar. Traditionally, however, this has entailed relatively harsh reaction conditions requiring: (1) periodate oxidation of the cis-diol group; followed by, (2) reductive amination. These harsh reaction conditions usually lead to denaturation of the protein.
It would be desirable to have a method for conjugating proteins which would not only proceed under relatively mild reactive conditions, but which would also take place in a discriminatory manner such that the reactive site of a protein would remain unmodified.